MHC antigens are glycoproteins expressed on the surface of cells, such as platelets, macrophages and lymphocytes. Functionally, these molecules play important roles in the presentation of foreign antigenic peptides to cytotoxic T cells. MHC molecules are divided into two types, Class I and Class II, based on their structure and function. MHC Class I genes encode glycoproteins which are expressed on the surface of almost all nucleated cells of the body. MHC Class I molecules are involved in activating cytotoxic T cells. MHC Class II genes encode glycoproteins expressed primarily on antigen presenting cells (macrophages, dendritic cells, B cells) where they present the processed antigen (e.g. viral or other foreign antigens) to the T helper cells. These antigens form one group of the so-called histocompatibility antigens.
MHC molecules in humans are referred to as Human leucocyte Antigen (HLA) molecules. Typing of the numerous HLA molecules present in humans has shown that individuals possess a particular ‘signature’ of HLA molecules present on their cells. HLA molecules are coded for in the human genome by a series of four gene loci. HLA Class I molecules are coded for by the A, B, C, E, F and G regions whereas the HLA Class II molecules are coded for by the DR, DQ, DP, DO and DM regions. The loci constituting the HLA molecules are highly polymorphic, and many alternate forms of the gene or alleles exist at each locus.
In normal immune responses self-MHC, e.g. HLA molecules are recognised by T cell receptors in vivo. The T cell receptors see the foreign antigen expressed as a small peptide in the context of a MHC Class I or class II molecule. This leads to production of appropriate antibody responses to the foreign antigen or destruction of the presenting cell depending on the presenting and T cells which are involved.
The immune System is however also able to identify and mount a challenge to non-self, i.e. foreign MHC molecules. Thus, when presented with a non-host MHC molecule, the immune system will react to destroy cells carrying the non-host MHC by normal immunological means, i.e. produce antibodies, activate the complement system etc. This is obviously undesirable when the cells carrying the non-host MHC, e.g. L are purposively introduced, for example, foreign cells or tissue, e.g. in a transplanted organ and presents a bar to the introduction of such cells.
Placing a ‘foreign’ (i.e. non-host) MHC molecule into an individual may result in the individual producing anti-MHC antibodies which will bind specifically to that MHC. Individuals may raise anti-MHC antibodies, and become “sensitized”, if they are exposed to a foreign MHC, i.e. during pregnancy, by blood transfusion, or by receiving an organ donation.
Pre-sensitization to MHC via transfusion, transplantation, or pregnancy can cause rapid rejection of transplanted tissue or poor platelet survival after transfusion. Therefore, testing for anti-MHC antibodies prior to tissue or organ transplantation is of great importance, as the presence in the recipient of anti-MHC antibodies which bind to donor MHC molecules (donor specific crossmatch) is predictive of a high risk of rejection of the transplanted tissue or organ. Thus, prior to transplantation, the donor tissue is typed for MHC molecules, and the recipient is typed for anti-MHC antibodies. Screening of potential transplant recipients for anti-HLA antibodies is an essential part of the pre-transplant monitoring carried out by tissue-typing laboratories.
Ideally, organs would be transplanted that are an ideal HLA match to the recipient. However, in view of the high number of HLA genes involved, up to hundred or more alleles for each, a perfect match is very difficult to obtain.
Post-transplant monitoring is also valuable to assess the level of anti-MHC antibodies which are being generated and hence the continued success or likely rejection of a transplant.
Anti-HLA antibody testing methods are known in the art, and include the screening of the blood or serum from the potential recipient against a panel of cello which are considered to present a representative selection of HLA antigens. This procedure can take up to 6 weeks. Such screening determines the panel reactivity (PR) for each sample, and gives an estimate of the degree of sensitisation against the panel of cello used for testing and can be related to the chance of a donor being suitable. The panel size may range from 25 to 100 different cells, and the larger the panels (50 to 100 different cells), the more reliable the results.
A more specific method is complement-dependent-lymphocytotoxicity (CDC) testing. In this method, the serum or blood of the potential recipient is tested against a panel of lymphocytes (or more specifically, the donor's lymphocytes) and the mixture is further incubated with complement factors. In order to measure the level of cytotoxicity, the viable and non-viable cells are distinguished using a dye. Therefore, this method is not without drawbacks, as the discrimination between living and dead cells can be subject to human error.
Another well known method is based on flow cytometry which is a method that allows the analysis of a large number of individual cells in a short time. The use of flow cytometric crossmatching of recipient serum against donor lymphocytes has shown it to be a more sensitive method of antibody detection than conventional cytotoxic crosshatch. Further, FACs (fluorescent activated cell sorting) screening of pooled cells can accurately and rapidly detect anti-MA antibodies. It has been shown (Harmer et al., Transpl. Int. (1993) 6: 277-280) that FACS can detect IgG antibodies which have not been detected by conventional screening methods.
However, these prior art methods are constrained due to the presence of molecules other than MA on the whole cells, and therefore other ‘non-HLA’ antibodies are detected. The recent use of ELISA (enzyme-linked-immunosorbent assay) employing purified class I and class II antigens from platelets and cell lines partially overcomes this problem.
In U.S. Pat. No. 5,948,627 (Lee et al.) the use of a plurality of microbeads presenting multiple purified HLA antigens from a cell population in order to test for anti-HLA antibodies is discussed.
Therefore, there is still a need for an assay for anti-MHC antibodies that detects single specific antibodies in a sample, in a quick and reliable manner. This will enable the dissection of component specificities and the detection of antibodies to rare MHC, e.g. HLA alleles in sera from highly sensitized patients. In brief, an assay is required that will precisely define the anti-MHC antibodies present in a sample, quickly, simply and reproducibly.